Immunosuppressant drugs such as sirolimus (also known as rapamycin), tacrolimus (also known as FK506), and cyclosporine (“CsA”) are effective for the treatment of organ or tissue rejection following transplant surgery, of graft versus host disease and of autoimmune diseases in humans. During immunosuppressant drug therapy, monitoring the blood concentration levels of the immunosuppressant is an important aspect of clinical care because insufficient drug levels lead to graft (organ or tissue) rejection and excessive levels lead to undesired side effects and toxicities. For instance, tacrolimus exhibits some toxicity similar to that of CsA, which includes nephrotoxicity, gastrointestinal tract complications and neurotoxicity. (See, Murthy, J. N., et al., Clinical Biochemistry, 31(8):613-617 (1998)). Blood levels of immunosuppressants are therefore measured so drug dosages can be adjusted to maintain the drug level at the appropriate concentration. Diagnostic assays for determination of immunosuppressant blood levels have thus found wide clinical use.
Tacrolimus is the generic name for a macrolide immunosuppressant produced by the bacterium Streptomyces tsukabaensis, in the soil (See, Inamura, N., et al., Transplantation, 45(1):206-209 (1988)). Tacrolimus has been used intravenously and orally for the prevention of organ rejection, particularly in patients receiving liver, kidney or bone marrow transplantation.
Cyclosporine is an immunosuppressive drug obtained from soil fungus (Tolypocladium inflatum). While primarily used to prevent organ rejection after transplant, CsA also has been used to treat other illnesses, such as aplastic anemia, or to prevent graft versus host disease. CsA's mode of action is to block T cell activation by preventing transcription of the IL-2 gene.
Tacrolimus has an in vivo potency 50-100 times greater than cyclosporine CsA (See, Murthy, J. N., et al., supra (1998)). The immunosuppressive effect of tacrolimus is similar to CsA and is thought to be through the selective inhibition of the generation of cytotoxic T cells. Id.
Other immunosuppressive drugs include sirolimus, everolimus, temsorolimus, zotarolimus, and mycophophenolic acid.
The primary target of sirolimus, everolimus (RAD-001), temsorolimus and zotarolimus is mTOR (mammalian Target of Rapamycin), a specific cell-cycle regulatory protein, the inhibition of which leads to suppression of cytokine-driven T-lymphocyte proliferation. Everolimus is used as an immunosuppressant to prevent rejection of organ transplants and as a cancer-suppressing drug. The contra-indication in the use of everolimus is a certain rise in cholesterol levels and therefore an increased cardio-vascular risk.
Mycophenolate mofetil (MMF) is an ester derivative of mycophenolic acid (MPA) and is approved as an immunosuppressant drug in renal transplant patients. The prodrug MMF is rapidly transformed in vivo to the active immunosuppressant MPA, which inhibits inosine monophosphate dehydrogenase 2. MMD thus suppresses the de novo synthesis of guanosine nucleotides, especially in T and B lymphocytes, and stops their proliferation.
A variety of different diagnostic immunoassays are commercially available for monitoring the blood concentrations of immunosuppressive drugs. While the immunoassays are available in a variety of formats, all use the binding of an antibody or binding protein (e.g. FKBP) to the immunosuppressant drug. A commonly-used prior art immunoassay is an assay which involves the binding of a first antibody to the immunosuppressant drug and the binding of labeled immunosuppressant (e.g., acridinylated sirolimus or tacrolimus) to the remaining free antibody binding sites, followed by quantitation through detection of the acridinium label.
Several of these immunoassays use organic solvents to extract the tacrolimus from whole blood samples. The organic solvent increases the equilibrium dissociation constant (KD) and/or lowers the functional activity of the antibody used in the assays. The reduced activity of the antibody leads to lower assay sensitivity and potentially lowers accuracy and robustness. Thus, the effectiveness of these immunoassays is affected by the particular extraction and denaturating solvent for the immunosuppressant that is used.
Likewise, the generation in vivo of metabolites to the immunosuppressive drug can impact assay results. Current literature suggests that the generation of CsA metabolites M17 (also known as AM1) and M1 (also known as AM9) can mask the concentration of active parent drug (CsA). Tacrolimus and sirolimus are known to form metabolites in vivo. The first generation major metabolites of tacrolimus are 13-O-demethylated tacrolimus (“M-I”), 31-O-demethylated tacrolimus (“M-II”), and 15-O-demethylated tacrolimus (“M-III”).
Immunoassay for the determination of MPA in serum (e.g., following MMF treatment) yields results that are higher than those obtained with the HPLC methods. This is believed due to the cross-reactivity of MPA metabolites with the monoclonal antibodies. MPA is metabolized mainly to a glucuronide derivative (MPAG), which is believed inactive. Recently, two other metabolites of MPA have been identified, including the acyl glucuronide of MPA.
Needless to say, the appropriate dosage of the immunosuppressant drug is critical for organ transplantation patients and needs to be accurately and reproducibly measured in the presence of metabolites.
Therefore, there is a need in the art for new immunoassays that provide accurate measures of drug analytes, e.g., immunosuppressives in the presence of their cross-reacting metabolites. It is an object of the disclosure to provide such immunoassays. It is a further object to provide such immunoassays which optimally avoid the problems that accompany modification of the extraction reagent composition to engender reduced cross-reactivity with metabolites. It is yet another object to provide immunoassays which optimally avoid the cost and labor considerations that accompany selection of an antibody or antibody optimization for reduced cross-reactivity with cross-reacting drug metabolites. Additional objects and embodiments will be apparent from the description herein.